The present invention is related to methods for the preparation of a polyalkoxylated nucleic acid molecule and a polyalkoxylated nucleic acid obtainable by such methods.
Attachment of polyalkoxylates such as polyalkylene glycols to drugs, such as small molecules, nucleic acid molecules, peptides, proteins and nanoparticles is widely used to increase the bioavailability, stability, safety, and efficacy of such drugs for therapeutic applications. Within the field of nucleic acid based drugs, aptamers and spiegelmers which are also referred to as mirror-image aptamers, are typically polyalkoxylated. Polyethylene glycol (abbr. PEG) is a typically used polyalkoxylate that has been approved by the Food and Drug Administration as part of drugs administered intravenously, orally and dermally.
In general, a polyalkoxylated nucleic acid molecule is prepared by a process that first assembles the nucleic acid molecule containing a reactive group on a solid phase. Such process is, for example, described in Hoffmann et al. (Hoffmann et. al, Current Protocols in Nucleic Acid Chemistry 2011, 4:4.46.1-4.46.30) and illustrated in FIG. 1A herein. After cleavage from solid phase the crude synthesis product can be purified by various processes such as Reversed Phase High Performance Liquid Chromatography (abbr. RP-HPLC), Ion Exchange Chromatography High Performance Liquid Chromatography (abbr. IEX-HPLC) and ultrafiltration or any combination thereof. Typically, such process consists of a combination of RP-HPLC or IEX-HPLC and ultrafiltration to yield a nucleic acid molecule comprising the desired nucleotide sequence and a reactive group for subsequent attachment of the polyalkoxylate. This reactive group can subsequently be reacted with a polyalkoxylate comprising a suitable reactive group capable of forming a covalent bond with the nucleic acid molecule and the reactive group provided by such nucleic acid molecule. After the conjugation reaction the thus obtained crude product is again purified by a process such as RP-HPLC, IEX-HPLC or ultrafiltration or any combination thereof.
The yield of a polyalkoxylation reaction depends on the purity of the nucleic acid molecule to be polyalkoxylated and on the reaction conditions itself. Because of that, typically the nucleic acid molecule to be polyalkoxylated is purified by HPLC and/or ultrafiltration before polyalkoxylation reaction. Standard conditions for polyalkoxylation reaction are as follows: The amino modified nucleic acid molecule is prepared in an aqueous solution and a base is added. Typically, the base is 100 mM sodium borate or sodium bicarbonate. Finally the polyalkoxylate-N-hydroxy succinimide ester (abbr. polyalkoxylate-NHS) is added dissolved in a water miscible organic solvent such as DMF or DMSO. The yield for a polyalkoxylated nucleic acid molecule is between 75 and 95% and largely depends on the equivalents of polyalkoxylate-NHS used. The yield suffers from the competing hydrolytic cleavage of the polyalkoxylate-NHS. Therefore, usually several equivalents of polyalkoxylate-NHS are added in several portions over time and the turnover is monitored by analytical methods.
Typically, therapeutic aptamers and spiegelmers consist of about 30 to 50 nucleotides (Keefe et. al., Nature Reviews, 2010, 9, 537; James, Encyclopedia of Analytical Chemistry, 2000, 4848). Although average stepwise coupling efficacy is close to 99%, a great number of nucleic acid molecules of truncated failure sequences of various lengths is accumulated during solid phase synthesis. For the separation of these truncated failure sequences from the desired full length nucleic acid species the properties of the finally coupled nucleotide, of a linker or modifier used in the attachment of the polyalkoxylate to the nucleic acid molecule and the nucleotide, respectively, or of the subsequently generated conjugate to the modifier is exploited as nucleic acid species having capped truncated sequences do not possess this building block. For example, in a 3′- to 5′-directed synthesis the 5′MMT- or 5′DMT-group of a C6-amino- or a C6-disulfide modifier can be used to achieve stronger interaction with IEX-HPLC or RP-HPLC-resins leading to later elution of full length species of the nucleic acid in comparisons to species having truncated failure species lacking the modification. Though the similar affinity effects can be used to separate unreacted truncated failure sequences of the polyalkoxylation reaction from the desired polyalkoxylated full length nucleic acid molecule, it is advantageous to remove the failure sequences prior to the final HPLC-purification as the resolution and efficiency of the chromatographic purification is influenced by the impurities. Purification by HPLC of polyalkoxylated nucleic acids such as, for example, aptamers and spiegelmers, however, is time consuming, laborious and involves expensive resins.
In light of the above, the production of polyalkoxylated nucleic acids involves numerous steps of synthesis and purification which are time and money consuming. Accordingly, there is a strong need to optimize the production of polyalkoxylated nucleic acids.